In analytical measurement procedures, fluid substances are analyzed among other substances. Two of the most common methods for the analysis of fluid substances are chromatography and capillary electrophoresis.
A chromatograph as well as a capillary electrophoresis device consist, in principle, of three units, viz. firstly the so called injector by means of which the substances containing the analyte, namely in most cases liquids, are injected into the system with a nanolitre or picolitre accuracy, secondly the separation column on which a spatial separation of the substances contained in the injected solution is carried out by means of physical or chemical interactions so that different substances will arrive at the end of the separation column at different times, and thirdly the detector at the exit of the separation column, said detector indicating the arrival of the individual substances contained in the solution.
The type of detection suitable for most classes of substances is the optical detection. When optical detection is carried out, light having a suitable wavelength is normally sent transversely through the separation column at the end of said separation column in an optical detector device and, consequently, it is sent through the solution to be analyzed. A certain percentage of the light is absorbed by the substances. The spectral position of the absorption peaks and their form make it possible to provide information on the nature and the concentration of the substances.
For various reasons, capillaries with inner diameters in the range of from a few micrometers up to approx. 100 micrometers are used as separation columns in the field of capillary electrophoresis and more and more also in the field of chromatography. When the light passes through the separation column transversely to the longitudinal direction thereof, the path of interaction between the light and the matter or rather the substance to be analyzed is very short so that the possible detection sensitivity is comparatively low.
In order to achieve a longer path of interaction between the light and the analyte-containing fluid in a fluid channel at the end of the separation column, it would be desirable to guide the light directly in the solution over a certain distance along the fluid channel in the separation column.
The aqueous solutions used most frequently as a solution have refractive indices of approx. n=1.33, whereas the typical capillary materials have refractive indices of at least n=1.47 in the case of quartz. Hence, the usual way of guiding the light by means of total internal reflection is impossible, since, for this purpose, the medium in which the light is to be guided must have a refractive index which is higher than that of the capillary material.
A solution used in connection with commercially available chromatography systems is the measure of increasing the path length of the light along which an interaction with the solution can take place by widening the capillary locally to a bubble through which the light is guided.
Another solution is to guide the light in the direction of the column within the fluid by total internal reflection at the boundary fluid/inner capillary wall. As has already been explained, the total internal reflection requires a chemical solution having a higher refractive index than the adjacent layer.
The technical publication A. Manz, D. J. Harrison, E. Verpoorte, H. M. Widmer: Planar chips technology for miniaturization of separation systems: a developing perspective in chemical monitoring. Advances in Chromatography 33(1993) 1-65 discloses the measure of guiding the light by means of the normal Fresnel reflections. This leads to significant losses, especially with small inner diameters of the capillaries and high wavelengths.
Another solution is to guide the light by total internal reflection in the wall of the separation column in order to increase the interaction in miniaturized separation systems. This method utilizes the absorption of the transversely attenuated field components in the solution to be analyzed. However, also this known system necessitates comparatively long interaction paths between the light and the solution to be analyzed, since only the weak evanescent waves of the light are absorbed.
To achieve total internal reflection, there are two methods which can be chosen: the first one is to use a chemical solution with a high refractive index, said refractive index being higher than that of the material of the capillary. This is achieved e.g. by the combinations of salt solutions with Teflon PFA or PFE coatings, as has been described in U.S. Pat. Nos. 4,009,382 as well as 3,954,341. The second one is the method of coating the inner wall of the capillary with a material having a low refractive index. In the first-mentioned case, there are two disadvantages: the aqueous salt solution is undesirable in many analytical measurements and the Teflon materials mentioned above are not sufficiently transparent at shorter UV wavelengths of the light used. When the second method which has been mentioned is used, the inner wall of the fused silica capillary has to be coated with a material having a low refractive index, this material being e.g. an amorphous fluoropolymere.